Serotonin transporter (SERT) is responsible for lowering serotonin (5-HT) levels in the vicinity of synapses that release this neurotransmitter. In this process, 5-HT is transported into the cell from which it was released, where it is available for repackaging into synaptic vesicles. Drugs that inhibit SERT increase the level of synaptic 5-HT and lengthen the duration of its action. These drugs include abused substances such as cocaine, and therapeutic drugs such as antidepressants. Some drugs that are substrates for SERT, such as 3,4-methylenedioxymethamphetamine (a.k.a. "ecstasy"), act through SERT to release 5-HT from neurons. From the profound behavioral consequences of these drugs, it is clear that regulation of SERT activity is likely to be a key event in normal brain physiology. An important new tool in understanding SERT regulation is a mutant form of the transporter found in patients with obsessive-compulsive disorder. This mutant is apparently activated in a constitutive fashion by a pathway normally involving cyclic GMP (cGMP). As part of the long-term goal to understand the structure and function of neurotransmitter transporters, this proposal outlines plans to investigate the activation of SERT by cGMP and the way that this process is disrupted in the mutant. The studies will approach these issues in three ways: 1) One approach will be to determine the pathway by which cGMP activates SERT. 2) The structural and mechanistic features of SERT that participate in the activation will be investigated. 3) The proposal also explores the nature of the SERT mutation to determine how it leads to constitutive activation of transport activity. If successful, this plan will identify the specific cGMP-dependent protein kinase responsible for SERT activation and will determine if the phosphorylation site is on SERT or another protein. The mechanism of activation through modification of SERT structure and conformation will also be investigated. The effect of environmental and behavioral influences on modification of SERT will be examined. All of these studies are directed to testing the hypothesis that production of cGMP activates a protein kinase that phosphorylates SERT with the consequent increase in turnover rate and ligand affinity. This hypothesis also predicts that the mutant form of SERT is defective as a substrate for a protein phosphatase.